Anti-braf-v600e recombinant rabbit monoclonal antibody and application thereof

Abstract

The application belongs to the technical field of immunochemistry, and particularly relates to an anti-BRAF-V600E recombinant rabbit monoclonal antibody and application thereof, which comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 5. The application also relates to a nucleotide sequence, a recombinant plasmid or an expression vector, a preparation method and application of the anti-BRAF-V600E recombinant rabbit monoclonal antibody in a BRAF-V600E protein detection method or device. The BRAF-V600E recombinant rabbit monoclonal antibody of the 135K5D1 clone of the application is good in specificity, strong in positive signal and the like, and is easier to score in IHC staining, and is more accurate in detection and distinction of cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunochemistry, and particularly relates to an anti-BRAF-V600E recombinant rabbit monoclonal antibody and application thereof, in particular, application in immunohistochemical detection. BACKGROUND

[0002] BRAF (serine / threonine-protein kinase B-Raf, proto-oncogene B-Raf) is a human gene, and the B-Raf protein encoded thereby is a core component of the intracellular signaling pathway (RAS-RAF-MEK-ERK), and participates in regulating the cell growth process by transmitting signals. Under normal circumstances, the activity of the B-Raf protein is strictly regulated by upstream signals, so as to ensure that the cell proliferation and differentiation are in a balanced state. The mutation of the BRAF gene can break this balance, and the V600E mutation is the most common oncogenic mutation. This change enables the B-Raf protein to acquire a constitutive activation ability, i.e., it can be in an active state continuously without the stimulation of upstream signals, and ultimately leads to cell over-proliferation, apoptosis resistance, and promotes the occurrence of cancer.

[0003] Based on the oncogenicity of the above mutation, the IHC detection of BRAF-V600E antibody can assist in the diagnosis and typing of various cancers, such as melanoma, papillary thyroid cancer, colorectal cancer, non-small cell lung cancer, etc., and can also guide the selection of targeted therapy programs. At present, there are several specific targeted drugs for BRAF-V600E mutation, such as vemurafenib, dabrafenib, etc., and the IHC detection result is a key prerequisite for judging whether the patient is suitable for such drugs. Only when the detection confirms the presence of BRAF-V600E mutation, the patient can benefit from targeted therapy; if it is wild type, the targeted drug is ineffective, and other treatment options need to be selected. In some tumors, the BRAF-V600E mutation status is closely related to the prognosis: in colorectal cancer, BRAF-V600E mutation usually indicates a worse prognosis; in papillary thyroid cancer, mutant patients are more likely to have lymph node metastasis and postoperative recurrence, and need closer follow-up.

[0004] The IHC detection of BRAF-V600E antibody has the advantages of rapidness, convenience and applicability to routine pathological specimens, and can be used as a preliminary screening method for the mutation status, and provides a reference for subsequent molecular detection, such as NGS, qPCR. Since BRAF-V600E is only a single amino acid substitution, the manufacture of antibodies targeting this mutation is extremely difficult, which also leads to certain limitations in the stability and specificity of its application in IHC, and the results of molecular detection often need to be combined for comprehensive judgment in clinic. SUMMARY

[0005] (1) Technical problems to be solved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a recombinant rabbit monoclonal antibody against BRAF-V600E that has wide applications and can accurately identify BRAF-V600E expression, and its application. The present invention also relates to the nucleotide sequence encoding the recombinant rabbit monoclonal antibody against BRAF-V600E, the recombinant plasmid or expression vector, the preparation method, and the application of the recombinant rabbit monoclonal antibody against BRAF-V600E in BRAF-V600E protein detection methods or devices.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] In a first aspect, the present invention provides a recombinant rabbit monoclonal antibody against BRAF-V600E, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

[0010] This recombinant rabbit monoclonal antibody against BRAF-V600E (BRAF-V600E rabbit-derived antibody) can be used for immunohistochemical detection, and can identify and detect the expression of BRAF-V600E protein on tumor cells or immune cells with high specificity and high sensitivity.

[0011] The anti-BRAF-V600E monoclonal antibody was obtained through recombinant expression in mammalian cells. Specifically, the anti-BRAF-V600E recombinant rabbit monoclonal antibody provided by this invention was produced through rabbit hybridoma fusion screening and eukaryotic expression in 293 cells. In preparing the anti-BRAF-V600E monoclonal antibody, the antigen used to immunize the rabbits (New Zealand White rabbits) was a synthetic polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1, and it was obtained through artificial chemical synthesis. After immunizing the rabbits, cell fusion and clonal screening were performed to obtain a positive hybridoma cell line that could efficiently secrete monoclonal antibodies. Molecular cloning technology was used to obtain the nucleotide sequences encoding the heavy chain and light chain amino acid sequences of the antibody. The nucleotide sequences were constructed on a eukaryotic expression vector, transfected into the 293 cell line using a transfection reagent, and the cell supernatant was collected and purified by protein A column affinity chromatography to obtain the rabbit monoclonal antibody. Immunohistochemical detection showed that the antibody specifically recognizes the BRAF-V600E protein.

[0012] The anti-BRAF-V600E monoclonal antibody can recognize recombinant BRAF-V600E antigen protein and BRAF-V600E molecules on tumor cells and immune cells; the anti-BRAF-V600E monoclonal antibody can also be used in immunohistochemical pathological diagnostic agents.

[0013] In a second aspect, the present application provides a coding gene for encoding the anti-BRAF-V600E recombinant rabbit monoclonal antibody.

[0014] Preferably, the coding gene comprises a DNA sequence as shown in SEQ ID NO: 2 or a sequence complementary thereto for encoding the heavy chain variable region of the anti-BRAF-V600E recombinant rabbit monoclonal antibody, and a DNA sequence as shown in SEQ ID NO: 3 or a sequence complementary thereto for encoding the light chain variable region of the anti-BRAF-V600E recombinant rabbit monoclonal antibody.

[0015] In a third aspect, the present application provides a nucleic acid molecule comprising a coding gene for encoding the anti-BRAF-V600E recombinant rabbit monoclonal antibody.

[0016] In a fourth aspect, the present application provides an expression vector or a recombinant plasmid comprising the nucleic acid molecule.

[0017] In a fifth aspect, the present application provides a host cell transformed or transfected with the expression vector or the recombinant plasmid.

[0018] In a sixth aspect, the present application provides a method for preparing the anti-BRAF-V600E recombinant rabbit monoclonal antibody, which comprises transforming or transfecting the host cell with the expression vector or the recombinant plasmid, culturing the transformed or transfected cell, collecting the supernatant of the cell and purifying to obtain the anti-BRAF-V600E recombinant rabbit monoclonal antibody.

[0019] In a seventh aspect, the anti-BRAF-V600E recombinant rabbit monoclonal antibody, the coding gene, the nucleic acid molecule, the expression vector or the recombinant plasmid are used for preparing a detection device for BRAF-V600E protein molecules. The detection device includes but is not limited to a kit, an antibody chip and the like.

[0020] In an eighth aspect, the present application further provides a BRAF-V600E detection kit comprising the anti-BRAF-V600E recombinant rabbit monoclonal antibody and an immunohistochemical detection reagent.

[0021] Preferably, the BRAF-V600E detection kit comprises the anti-BRAF-V600E recombinant rabbit monoclonal antibody, a horseradish peroxidase-labeled secondary antibody, an ethylenediaminetetraacetic acid repair solution, a catalase blocking solution, a 3,3'-diaminobenzidine concentrate solution, a 3,3'-diaminobenzidine buffer solution, hematoxylin and a bluing solution.

[0022] In the immunohistochemical detection, the detection steps include deparaffinization, antigen repair, endogenous peroxidase inactivation, blocking, first antibody incubation, second antibody incubation, 3,3'-diaminobenzidine (DAB) color development, counterstaining, dehydration, mounting and microscopic examination.

[0023] (III) Beneficial Effects

[0024] The anti-BRAF-V600E recombinant rabbit monoclonal antibody provided by the application has high specificity and high sensitivity in binding with a BRAF-V600E protein molecule, can specifically recognize and detect the expression of the BRAF-V600E protein on cells, is positively highly expressed in the detection of the BRAF-V600E protein, and is found through immunohistochemical detection of various different tissues that the antibody can be applied to the detection and screening fields such as immunohistochemistry (IHC), indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, antibody chip preparation, and flow cytometry, and is beneficial to obtaining accurate evaluation and detection results. The BRAF-V600E recombinant rabbit monoclonal antibody of the 135K5D1 clone of the application is easier to score in IHC staining and more accurate in distinguishing cancer due to its good specificity and strong positive signal. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The figure is the immunohistochemical detection result of the 135K5D1 anti-BRAF-V600E monoclonal antibody and a commercially available antibody in the preparation of the application in thyroid papillary carcinoma, and the concentration of the 135K5D1 anti-BRAF-V600E monoclonal antibody is 1 μg / mL; wherein a is the immunohistochemical detection result of the BRAF-V600E antibody of the 135K5D1 clone in thyroid papillary carcinoma, and b is the immunohistochemical detection result of the commercially available antibody in thyroid papillary carcinoma (a and b have the same magnification);

[0026] Figure 2 The figure is a statistical graph of titer detection of the 135K5D1 anti-BRAF-V600E monoclonal antibody and a commercially available antibody in the preparation of the application at 8 gradient concentrations;

[0027] Figure 3 The figure is the detection result of Western blotting of the 135K5D1 anti-BRAF-V600E monoclonal antibody as a primary antibody, which verifies the recognition ability of the antibody to the BRAF-V600E protein. DETAILED DESCRIPTION

[0028] To further illustrate the technical means adopted by the present application and its effects, the present application is further described below in conjunction with examples and drawings. It can be understood that the specific embodiments described herein are merely intended to explain the present application, but not to limit the present application.

[0029] Unless otherwise specified in the examples, the techniques or conditions are in accordance with those described in the literature in the art, or in accordance with the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be commercially available through regular channels. Human tissue samples are formalin-fixed, paraffin-embedded human tissue samples, which have been pathologically confirmed and have patient informed consent.

[0030] Example 1

[0031] This example is the preparation and screening of anti-BRAF-V600E recombinant rabbit monoclonal antibody, which includes the following steps:

[0032] (1) Antigen preparation

[0033] The specific sequence of the BRAF-V600E antigen is shown in SEQ ID NO: 1 as follows.

[0034] SEQ ID NO: 1 is KIGDFGLATEKSRWSGSH.

[0035] The above polypeptide sequence is selected by analyzing the sequence of BRAF-V600E molecule, according to the structure, antigenicity, hydrophobicity of constituent amino acids and secondary structure of BRAF-V600E protein molecule. The polypeptide of the sequence shown in SEQ ID NO: 1 is artificially synthesized, and the synthesized polypeptide is used as an antigen for immunizing rabbits. When immunizing, the polypeptide of the sequence shown in SEQ ID NO: 1 is coupled with keyhole limpet hemocyanin (KLH) after immunizing rabbits as BRAF-V600E antigen.

[0036] (2) Immunization

[0037] The BRAF-V600E antigen obtained in step (1) is mixed with complete Freund's adjuvant (volume ratio 1:1) and emulsified, and a plurality of New Zealand white rabbits are immunized by subcutaneous injection. After two weeks, the BRAF-V600E antigen containing the above sequence (polypeptide shown in SEQ ID NO: 1) is emulsified with incomplete Freund's adjuvant (volume ratio 1:1) for the second and third immunization. After three immunizations, blood is taken to determine the serum titer by ELISA method with gradient dilution; the rabbit with the highest antibody titer to the SEQ ID NO: 1 antigen is selected for the next cell fusion.

[0038] (3) Cell fusion

[0039] Prepare mouse-derived sp2 / 0 myeloma cells in advance, so that the sp2 / 0 myeloma cells are in the logarithmic growth phase at the time of fusion. Take the spleen of the immunized rabbit and prepare a lymphocyte single cell suspension; mix the rabbit spleen lymphocytes with the myeloma cells, add 50% PEG1500 dropwise, add IMDM medium, centrifuge and discard the supernatant, then add HAT medium and mix gently, make up to 800 mL, then dispense into 96-well plates, and place in a 37°C, 5% CO2 incubator for culture. After 6-9 days of fusion, observe the state of the fused cells in the 96-well plates, replace the liquid with HT, and continue to culture in a 37°C, 5% CO2 incubator.

[0040] (4) Screening and cloning

[0041] After 7-10 days of fusion, perform ELISA testing using the BRAF-V600E antigen (SEQ ID NO: 1) to screen the cloned cells. Label the corresponding cell strain number, and perform limited dilution on the positive well cells until the ELISA determination 96-well plate results are positive. Select a single monoclonal stable strain with high positive value to obtain a hybridoma cell strain that secretes a specific monoclonal antibody, and record it as 135K5D1.

[0042] (5) Antibody sequencing of the screened hybridoma cell strain

[0043] According to the instructions of the TriZol RNA extraction reagent, total RNA is isolated from the 135K5D1 hybridoma cells, and according to the instructions of the TIANScript first-strand cDNA synthesis kit, the total RNA is reverse transcribed into cDNA. The antibody heavy chain variable region and antibody light chain variable region nucleotide sequences are obtained by amplification using specific primers (heavy chain variable region primers, VH-F: AGACTGGGCTGCGCTGGCTTC, VH-R GTGAGGGTGCCCGAG; light chain variable region primers: VK-F ATGGACAYGAGGGCCCCCACTC, VK-R: GGTGGGAAGATGAGGACAGTAGG). Then, the antibody heavy chain variable region and antibody light chain variable region nucleotide sequences are cloned into a eukaryotic expression vector (InvivoGen, pfuse-rchg, pfuse2-rclk1) for cell transfection.

[0044] (6) Cell transfection and screening

[0045] Prepare 293 cells to be transfected in advance, centrifuge and replace with fresh culture medium, then place in 24-well plates, 1.5 ml per well, density 3x10 6 6 / ml.

[0046] The eukaryotic expression vector is mixed with polyethyleneimine (PEI) at a mass ratio of 1:6, then added to the prepared 293 cells, and cultured in a 37°C, 5% CO2 shaker. After 3-5 days of culture, the transfected cell supernatant is detected by ELISA with the corresponding antigen to screen positive wells, and the cell supernatant of the positive wells is further detected by immunohistochemical method. If the immunohistochemical method is positive, it is confirmed that the detected antibody sequence is correct.

[0047] (7) Preparation and purification of cell supernatant monoclonal antibody

[0048] The positive expression vector is subjected to large-scale cell transfection, and after 3-5 days of continuous culture, the cell suspension is collected, centrifuged, and the supernatant is obtained. The affinity chromatography method is used to purify the supernatant using protein A to obtain an antibody with a purity of > 95%. The monoclonal antibody concentration after purification is determined, divided, and stored in a 4-8°C refrigerator.

[0049] Finally, the heavy chain variable region nucleotide sequence of the 135K5D1 anti-BRAF-V600E recombinant rabbit monoclonal antibody is shown in SEQ ID NO: 2, and the light chain variable region nucleotide sequence of the anti-BRAF-V600E recombinant rabbit monoclonal antibody is shown in SEQ ID NO: 3.

[0050] The specific sequences of SEQ ID NO: 2 and SEQ ID NO: 3 are as follows:

[0051] SEQ ID NO: 2 is:

[0052] cagtcgctggaggagtccgggggtcgcctggtcacgcctgggacacccctgatactcacctgcacagtctctggattctccctcagtagcgattcaataaactgggtccgccaggctccagggaaggggctggaatggatcggaaccattagtaatgttgggatcacatactacgcgacctgggcgaaaggccgattcaccatctccaaaacctcgtcgaccacggtggatctgaaaatgaccagtccgacaaccgaggacacggccacctatttgtgtgtcagacggggtgactggttgtggggccaaggcaccctggtcaccgtctcctca;

[0053] SEQ ID NO: 3 is:

[0054] gccgccgtgctgacccagactccatctcccgtgtctgcagctgtgggaggcacagtcagcatcagttgccagtccagtaagagtgtttatagtgacaactggttaggctggtatcagcagaaaccagggcagcctcccaagctcctgatctacaaggcttccactctggcatctggggtcccatcgcggttcaaaggcagtggatctgggacacaattcactctcaccatcagcgccgtgcagtgtgacgatgctgccacttactactgtgcaggcggttatagtggtggtagtgataaaagtagtttcggcggagggaccgaggtggtggtcaaa.

[0055] The obtained base sequence is translated into amino acid sequence, analyzed, and the amino acid sequence of the heavy chain variable region of the anti-BRAF-V600E recombinant rabbit monoclonal antibody is shown as SEQ ID NO: 4, and the amino acid sequence of the light chain variable region of the anti-BRAF-V600E recombinant rabbit monoclonal antibody is shown as SEQ ID NO: 5.

[0056] The specific sequences of SEQ ID NO: 4 and SEQ ID NO: 5 are as follows:

[0057] SEQ ID NO: 4 is:

[0058] QSLEESGGRLVTPGTPLILTCTVSGFSLSSDSINWVRQAPGKGLEWIGTISNVGITYYATWAKGRFTISKTSSTTVDLKMTSPTTEDTATYLCVRRGDWLWGQGTLVTVSS;

[0059] SEQ ID NO: 5 is:

[0060] AAVLTQTPSPVSAAVGGTVSISCQSSKSVYSDNWLGWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISAVQCDDAATYYCAGGYSGGSDKSSFGGGTEVVVK.

[0061] Example 2

[0062] This example is an immunohistochemical detection of the anti-BRAF-V600E recombinant rabbit monoclonal antibody as a primary antibody, and the method is as follows:

[0063] (1) Sample slice preparation: The paraffin-embedded thyroid papillary carcinoma tissue slice after formalin fixation is baked in a 60°C incubator for 1-2 hours, and is stored for use;

[0064] (2) Slice deparaffinization: The paraffin slice is first placed in fresh xylene for deparaffinization, and is soaked for 2 times, each time for 10 minutes;

[0065] (3) Slice hydration: Hydration is performed by soaking in anhydrous ethanol, anhydrous ethanol, 95% ethanol, 85% ethanol, and 70% ethanol for 5 minutes, and then pure water is used for flushing for 2 times, each time for 3 minutes;

[0066] (4) Antigen repair: High-temperature heat repair method is recommended for repair for 3 minutes (if an automatic repair instrument is used, 98°C high-temperature repair can be set for 20 minutes), and after the slice is naturally cooled to room temperature, the tissue to be tested is circled with an immunohistochemical pen, and pure water is used for flushing for 2 times, each time for 3 minutes;

[0067] (5) Endogenous peroxidase inactivation: An appropriate amount of endogenous peroxidase blocking agent is dropped to completely cover the tissue, and after incubation at room temperature for 10 minutes, pure water is used for flushing for 2 times, each time for 3 minutes, and phosphate buffered saline (PBST) is used for flushing once;

[0068] (6) Primary antibody incubation: 100 μL of 1 μg / mL 135K5D1 anti-BRAF-V600E recombinant rabbit monoclonal antibody is added to completely cover the tissue, and the slice is incubated in a 37°C incubator for 1 hour, and PBST is used for flushing for 3 times, each time for 5 minutes;

[0069] (7) Secondary antibody incubation: Secondary antibody incubation is performed according to the instructions of the DAB staining liquid kit used for staining, and after the incubation is completed, the slice is flushed with PBST for 3 times, each time for 5 minutes, and pure water is used for flushing once;

[0070] (8) DAB color development: DAB color development liquid is prepared according to the instructions of the DAB staining liquid kit used, and an appropriate amount of prepared DAB color development liquid is dropped to completely cover the tissue, and the staining is terminated when the color does not deepen, and the slice is flushed with pure water for 3 times;

[0071] (9) Hematoxylin restaining: The slice is restained according to the operation steps and suggestions of the hematoxylin manufacturer, and is flushed with PBST or tap water to return to blue;

[0072] (10) Dehydration and transparency: The slice is soaked in 70%, 85%, 95%, and 100% gradient alcohol for 3 minutes each time, and is transparentized in xylene for 2 times, each time for 5 minutes;

[0073] (11) Mounting: The sample is mounted with neutral balsam.

[0074] By Figure 1 As can be seen from the results, the BRAF-V600E protein is specifically stained in the cytoplasm of human thyroid papillary tumor tissues, and the staining effect of the BRAF-V600E recombinant rabbit monoclonal antibody of the 135K5D1 clone is better than that of the commercially available BRAF-V600E antibody, and the staining color is darker. It is proved that the BRAF-V600E recombinant rabbit monoclonal antibody of the 135K5D1 clone of the application is easy to evaluate and has high recognition due to its good specificity and strong positive signal, and is more accurate for detecting and distinguishing cancer.

[0075] Example 3

[0076] This example is the determination of the affinity of the 135K5D1 anti-BRAF-V600E recombinant rabbit monoclonal antibody, and the determination method is as follows:

[0077] (1) Take out the labeled BRAF-V600E polypeptide (SEQ ID NO: 1) from 4°C and restore to room temperature. Dilute to a concentration of 1 μg / ml, add 100 μL / well to the 96-well enzyme-labeled plate, and incubate at 4°C overnight, then block with 2% bovine serum albumin (BSA) at 4°C overnight;

[0078] (2) Dilute the 135K5D1 clone BRAF-V600E recombinant rabbit monoclonal antibody to an initial concentration of 0.5 μg / mL, and sequentially perform 2-fold gradient dilution, a total of 8 concentration gradients for comparison;

[0079] (3) Add the diluted anti-BRAF-V600E recombinant rabbit monoclonal antibody to the 96-well enzyme-labeled plate with polypeptide at 100 μL / well, cover with a sealing film, and incubate at 37°C for 1 h to make the reaction reach equilibrium;

[0080] (4) After the reaction is completed, take out the enzyme-labeled plate, discard the liquid, rinse with purified water for 5 times, and pat dry the water;

[0081] (5) Dilute the horseradish peroxidase (HRP) labeled sheep anti-rabbit IgG according to the instructions, add 100 μL / well to the enzyme-labeled plate, and incubate at 37°C for 1 h to make the reaction reach equilibrium;

[0082] (6) After the reaction is completed, take out the enzyme-labeled plate, discard the liquid, rinse with purified water for 5 times, and pat dry the water;

[0083] (7) Add 3,3',5,5'-tetramethylbenzidine (TMB) color developing liquid at 100 μL / well, and react at room temperature for 6 minutes;

[0084] (8) After the reaction is completed, add 2M H2SO4 at 50 μL / well to terminate the color development;

[0085] (9) Read OD value at 450nm on the microplate reader, organize data, and analyze results as shown in Table 4. Figure 2

[0086] The results show that the anti-BRAF-V600E recombinant rabbit monoclonal antibody 135K5D1 of the application has strong affinity to BRAF-V600E protein molecules and high sensitivity, and can achieve high OD value under lower antibody concentration, thereby saving experimental and detection costs.

[0087] Example 4

[0088] This example is the Western blotting detection of the anti-BRAF-V600E recombinant rabbit monoclonal antibody 135K5D1 as primary antibody, and the method is as follows:

[0089] (1) Select polyvinylidene fluoride (PVDF) membranes of Braf wild type expression cell strain, Braf mutant type expression cell strain, Raji, Hela, A549, T47D, MDA-MB-453 cell lysate for activation, methanol activation for 1 min, and then wash the membrane with pure water for 2 times and then wash with TBST for 3 times; blocking: place the membrane in the blocking solution prepared by 5% bovine serum albumin (BSA), and shake at room temperature for 2 h; the TBST is a commonly used washing buffer suitable for experiments such as Western blotting. It contains three basic components: Tris buffer, salt (usually sodium chloride), and surfactant Tween-20.

[0090] (2) Primary antibody incubation: dilute the 135K5D1 antibody to a concentration of 0.5 μg / mL, and place the blocked membrane in the corresponding diluted antibody, and incubate overnight at 4°C;

[0091] (3) Take out the membrane and wash it in TBST solution for 3 times (2x5min+1x10min);

[0092] (4) Secondary antibody incubation: dilute HRP-anti-rabbit IgG with FG solution at a ratio of 1:5000, mix well, and then add to the membrane strip, and shake at room temperature for 1 h;

[0093] (5) Take out the membrane strip and wash it in TBST solution for 4 times (3x5min+1x8min);

[0094] (6) Substrate: mix equal amounts of Luminol / enhancer solution and Peroxide solution diluted 5 times with pure water in the same container, add the membrane strip, and incubate for 2 min;

[0095] ​(7) Exposure: the film is placed in the dark box, according to the intensity of fluorescence X-ray film for different time exposure; then according to the order of 1min development, washing, 1min fixing operation, finally washing and drying; results as shown in Figure 3

[0096] The theoretical molecular weight of BRAF-V600E is about 84kDa, and the actual measured molecular weight is greater than the theoretical value due to the presence of phosphorylation, glycosylation modification and the like of BRAF-V600E protein. Figure 3 Braf-600V represents the wild type Braf protein expression cell strain lysate, Braf-600E represents the mutant Braf protein expression cell strain lysate, Raji represents the mouse embryonic fibroblast cell lysate, Hela represents the mouse embryonic fibroblast cell lysate, A549 represents the mouse embryonic fibroblast cell lysate, T47D represents the mouse embryonic fibroblast cell lysate, and MDA-MB-453 represents the mouse embryonic fibroblast cell lysate. Figure 3 It can be seen from the results that in the lane, the 135K5D1 anti-BRAF-V600E recombinant rabbit monoclonal antibody can specifically recognize the BRAF-V600E protein in the mutant protein expression cell, and the wild type protein in the wild type expression cell strain and other tumor cell strains cannot be recognized, which shows that the 135K5D1 clone BRAF-V600E recombinant rabbit monoclonal antibody of the application can specifically recognize the BRAF-V600E protein.

[0097] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.​

Claims

1. A recombinant rabbit monoclonal antibody against BRAF-V600E, characterized in that, It includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

5.

2. A gene encoding a gene, characterized in that, Used to encode the recombinant rabbit monoclonal antibody against BRAF-V600E as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, It includes: DNA sequences as shown in SEQ ID NO:2 are used to encode the heavy chain variable region of the anti-BRAF-V600E recombinant rabbit monoclonal antibody, and DNA sequences as shown in SEQ ID NO:3 are used to encode the light chain variable region of the anti-BRAF-V600E recombinant rabbit monoclonal antibody.

4. A nucleic acid molecule, characterized in that, It contains the coding gene as described in claim 2 or 3.

5. An expression vector or recombinant plasmid, characterized in that, It contains the nucleic acid molecule as described in claim 4.

6. Transform or transfect the host cell of the expression vector or recombinant plasmid as described in claim 5.

7. A method for preparing a recombinant rabbit monoclonal antibody against BRAF-V600E, characterized in that, The expression vector or recombinant plasmid described in claim 5 is used to transform or transfect the host cells described in claim 6. The transformed or transfected cells are cultured, and the cell supernatant is collected and purified to obtain the anti-BRAF-V600E recombinant rabbit monoclonal antibody.

8. The use of the anti-BRAF-V600E recombinant rabbit monoclonal antibody of claim 1, the encoding gene of claim 2 or 3, the nucleic acid molecule of claim 4, the expression vector or recombinant plasmid of claim 5, and the host cell of claim 6 in the preparation of a BRAF-V600E detection device.

9. A BRAF-V600E detection kit, characterized in that, It includes the anti-BRAF-V600E recombinant rabbit monoclonal antibody and immunohistochemical detection reagent as described in claim 1.

10. The BRAF-V600E detection kit according to claim 9, characterized in that, It includes: the anti-BRAF-V600E recombinant rabbit monoclonal antibody as described in claim 1, horseradish peroxidase-labeled secondary antibody, EDTA retrieval solution, catalase blocking solution, 3,3'-diaminophenylhydrazine concentrate, 3,3'-diaminophenylhydrazine buffer, hematoxylin and blueing solution.

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